Human parainfluenza virus type 3 (HPIV3) is a common cause of serious lower respiratory tract infection in infants and children less than one year of age. It is second only to respiratory syncytial virus (RSV) as a leading cause of hospitalization for viral lower respiratory tract disease in this age group (Collins et al., p. 1205-1243. In B. N. Fields (Knipe et al., eds), Fields Virology, 3rd ed, vol. 1. Lippincott-Raven Publishers, Philadelphia, 1996; Crowe et al., Vaccine 13:415-421, 1995; Marx et al., J. Infect. Dis. 176:1423-1427, 1997). Infections by this virus results in substantial morbidity in children less than 3 years of age. HPIV1 and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup) and also can cause severe pneumonia and bronchiolitis (Collins et al., 3rd ed. In xe2x80x9cFields Virology,xe2x80x9d B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melinck, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996). In a long term study over a 20-year period, HPIV1, HPIV2, and HPIV3 were identified as etiologic agents for 6.0, 3.2, and 11.5%, respectively, of hospitalizations for respiratory tract disease accounting in total for 18% of the hospitalizations, and, for this reason, there is a need for an effective vaccine (Murphy et al., Virus Res 11, 1-15, 1988). The parainfluenza viruses have also been identified in a significant proportion of cases of virally-induced middle ear effusions in children with otitis media (Heikkinen et al., N Engl J Med 340:260-4, 1999). Thus, there is a need to produce a vaccine against these viruses that can prevent the serious lower respiratory tract disease and the otitis media that accompanies these HPIV infections.
Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV strain, nor for ameliorating HPIV related illnesses. To date, only two live attenuated PIV vaccine candidates have received particular attention. One of these candidates is a bovine PIV (BPIV3) strain that is antigenically related to HPIV3 and which has been shown to protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in human infants and children (Karron et al., J. Inf. Dis. 171:1107-14 (1995a); Karron et al., J. Inf. Dis. 172:1445-1450, (1995b)). A second PIV3 vaccine candidate, JS cp45 is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (arron et al., (1995b), supra; Belshe et al., J. Med. Virol. 10:235-42 (1982)). This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold-adaptation (ca), and attenuation (att) phenotypes which are stable after viral replication in vivo. The cp45 virus is protective against human PIV3 challenge in experimental animals and is attenuated, genetically stable, and immunogenic in seronegative human infants and children (Hall et al., Virus Res. 22:173-184 (1992); Karron et al., (1995b), supra).
To facilitate development of PIV vaccine candidates, recombinant DNA technology has recently made it possible to recover infectious negative-stranded RNA viruses from cDNA (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89 (1996); Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), rinderpest virus, simian virus 5 (SV5), Newcastle disease virus (NDV), and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81 (1995); Radecke et al., EMBO J. 14:5773-5784 (1995); Schnell et al., EMBO J. 13:4195-203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92 (1995); Hoffman et al., J. Virol. 71:4272-4277 (1997); Kato et al., Genes to Cells 1:569-579 (1996), Roberts et al., Virology 247(1), 1-6 (1998); Baron et al., J. Virol. 71:1265-1271 (1997); International Publication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567 (1995); U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding to published International Application No. WO 98/02530 and priority U.S. Provisional Application Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filed Jul. 15, 1996); Juhasz et al., J. Virol. 71(8):5814-5819 (1997); He et al. Virology 237:249-260 (1997); Baron et al. J. Virol. 71:1265-1271 (1997); Whitehead et al., Virology 247(2):232-9 (1998a); Whitehead et al., J. Virol. 72(5):4467-4471 (1998b); Peeters et al. J. Virol. 73:5001-5009, 1999; Jin et al. Virology 251:206-214 (1998); Bucholz et al. J. Virol. 73:251-259 (1999); and Whitehead et al., J. Virol. 73:(4)3438-3442 (1999), each incorporated herein by reference in its entirety for all purposes).
In more specific regard to the instant invention, a method for producing HPIV with a wt phenotype from cDNA was recently developed for recovery of infectious, recombinant PIV3 JS strain (see, e.g., Durbin et al., Virology 235:323-332, 1997; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998; U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), and U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997, each incorporated herein by reference). In addition, these disclosures allow for genetic manipulation of cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, e.g., which mutations in the HPIV3 cp45 virus specify its ts, ca and att phenotypes, and which gene(s) of BPIV3 specify its attenuation phenotype. Additionally, these disclosures render it feasible to construct novel PIV vaccine candidates and to evaluate their level of attenuation, immunogenicity and phenotypic stability.
Thus, infectious wild type recombinant PIV3 (r)PIV3, as well as a number of ts derivatives, have now been recovered from cDNA, and reverse genetics systems have been used to generate infectious virus bearing defined attenuating mutations and to study the genetic basis of attenuation of existing vaccine viruses. For example, the three amino acid substitutions found in the L gene of cp45, singularly or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and attenuating mutations are present in other regions of the PlV3cp45. In addition a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3 full-length cDNA that contains the three attenuating mutations in L. The recombinant chimeric virus derived from this cDNA is designated rPIV3-1.cp45L (Skiadopoulos et al., J Virol 72:1762-8, 1998; Tao et al., J Virol 72:2955-2961, 1998; Tao et al., Vaccine 17:1100-1108, 1999, incorporated herein by reference). rPIV3-1.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with PIV1. A recombinant chimeric virus, designated rPIV3-1.cp45, has been produced that contains 13 of the 15 cp45 mutations, i.e., excluding the mutations in HN and F, and is highly attenuated in the upper and lower respiratory tract of hamsters (Skiadopoulos et al., Vaccine In press, 1999).
Despite these numerous advances toward development of effective vaccine agents against different PIV groups, there remains a clear need in the art for additional tools and methods to engineer safe and effective vaccines to alleviate the serious health problems attributable to PIV, particularly illnesses among infants and children due to infection by HPIV3. Among the remaining challenges in this context is the need for additional tools to generate suitably attenuated, immunogenic and genetically stable vaccine candidates for use in diverse clinical settings. To facilitate these goals, existing methods for identifying and incorporating attenuating mutations into recombinant vaccine strains must be expanded. Surprisingly, the present invention fulfills this need and provides additional advantages as described hereinbelow.
The present invention provides novel tools and methods for introducing defined, predetermined structural and phenotypic changes into infectious PIV. In one embodiment of the invention, an isolated polynucleotide molecule is provided which comprises an operably linked transcriptional promoter, a polynucleotide sequence encoding a PIV genome or antigenome, and a transcriptional terminator. The genome or antigenome incorporates a knock out mutation that reduces or ablates expression of one or more of the C, D, and/or V gene(s), or a mutation that deletes all or a portion of one or more of the C, D, and/or V ORFs.
In preferred aspects of the invention, expression of one or more of the C, D, and/or V ORFs is reduced or ablated by modifying the PIV genome or antigenome to incorporate a mutation that changes the start codon from M to T for the C ORF or one or more stop codons. Alternatively, one or more of the C, D, and/or V ORFs is deleted in whole or in part or modified by other mutations such as point mutations to render the corresponding protein(s) partially or entirely non-functional or to disrupt protein expression altogether. Alternatively, mutations can be made in the editing site that prevent editing and ablate expression of proteins accessed by RNA editing (Kato et al., EMBO 16:578-587, 1997 and Schneider et al., Virology227:314-322, 1997).
The recombinant PIV of the invention having mutations in C, D, and/or V possess highly desirable phenotypic characteristics for vaccine development. The above identified modifications in the recombinant genome or antigenome specifies one or more desired phenotypic changes in the resulting virus or subviral particle. Vaccine candidates are thus generated that exhibit one or more characteristics identified as (i) a change in growth properties in cell culture, (ii) attenuation in the upper or lower respiratory tract of mammalian hosts, (iii) a change in viral plaque size, (iv) a change in cytopathic effect, and (v) a change in immunogenicity.
In exemplary PIV recombinants described herein, desired phenotypic changes include attenuation of viral growth in vitro and/or in mammalian hosts compared to growth of a corresponding wild-type or mutant parental PIV strain. In more detailed aspects, viral growth in cell culture may be attenuated by approximately 5-10-fold attributable to the knock out or gene (or genome segment) deletion mutations. Viral attenuation in the upper and/or lower respiratory tract of mammalian hosts is preferably attenuated approximately 10-100 fold, and sometimes 100-1,000 fold or greater to facilitate vaccine development. At the same time, recombinant PIV of the invention have immunogenic characteristics that stimulate a protective immune response against wt PIV challenge in both the upper and lower respiratory tracts of mammalian hosts.
The PIV genome or antigenome bearing a C, D, and/or V knockout mutation(s) can be a human or nonhuman PIV sequence, or a recombinantly modified version thereof. In one embodiment, the polynucleotide sequence encodes a chimeric genome or antigenome comprising a human PIV sequence recombinantly joined with a nonhuman PIV sequence, such as a gene or gene segment from bovine PIV (BPIV) (see, e.g., U.S. Provisional Application No. 60/143,134, filed by Bailey et al. on Jul. 9, 1999, incorporated herein by reference). In additional examples, the polynucleotide encodes a chimera of sequences from a nonhuman PIV and at least one other PIV of human or nonhuman origin.
In other embodiments, the invention provides an isolated infectious PIV particle comprising a recombinant PIV (rPIV) genome or antigenome incorporating a C, D, and/or V knockout mutation(s). The isolated infectious PIV particle can be a viral or subviral particle. As used herein, subviral particle refers to any infectious PIV particle which lacks a structural element, eg., a gene segment, gene, protein, or protein functional domain, which is present in a complete virus (eg., an assembled virion including a complete genome or antigenome, nucleocapsid and envelope). Thus, one example of a subviral particle of the invention is an infectious nucleocapsid containing a genome or antigenome, and the products of N, P, and L genes. Other subviral particles are produced by partial or complete deletions or substitutions of non-essential genes, genome segments, and/or partial or complete gene products (eg., F, HN, M, or C), among other non-essential genomic and structural elements.
In combination with the phenotypic effects provided in recombinant PIV bearing a C, D, and/or V deletion or knock out mutation(s), it is often desirable to adjust the attenuation and immunogenic phenotype by introducing additional mutations that increase or decrease attenuation and/or modulate immunogenic activity of the recombinant virus. Thus, candidate vaccine strains can be further attenuated by incorporation of at least one, and preferably two or more different attenuating mutations, for example mutations identified in a biologically derived PIV mutant or identified emprically using recombinant mini-replicons, recombinant virus, or biologically-derived virus. Preferred mutant PIV strains in this context are cold passaged (cp), cold adapted (ca), host range restricted (hr), small plaque (sp), and/or temperature sensitive (ts) mutants, for example the JS HPIV3 cp 45 mutant strain. In exemplary embodiments, one or more attenuating mutations occur in the polymerase L protein, e.g., at a position corresponding to Tyr942, Leu992, or Thr1558 of JS cp45. Alternatively, attenuating mutations in the N protein may be selected and incorporated in a C, D, and/or V deletion or knock out mutant, for example which encode amino acid substitution(s) at a position corresponding to residues Val96 or Ser389 of JS cp45. Alternative or additional mutations may encode amino acid substitution(s) in the C protein, e.g., at a position corresponding to Ile96 of JS cp45. Yet additional mutations for adjusting attenuation of a C, D, and/or V deletion or knock out mutant of the invention are found in the F protein, e.g., at a position corresponding to Ile420 or Ala450 of JS cp45, and in the HN protein, e.g., at a position corresponding to residue Val384 of JS cp45 (Skiadopoulos et al., J. Virol. 73:1374-1381, 1999; Skiadopoulos et al., J Virol. 73:1374-1381, 1999, each incorporated herein by reference).
Attenuating mutations from biologically derived PIV mutants for incorporation into C, D, and/or V deletion or knock out mutants of the invention also include mutations in noncoding portions of the PIV genome or antigenome, for example in a 3xe2x80x2 leader sequence. Exemplary mutations in this context may be engineered at a position in the 3xe2x80x2 leader of a recombinant virus at a position corresponding to nucleotide 23, 24, 28, or 45 of JS cp45 (Skiadopoulos et al., J. Virol. 73:1374-1381, 1999, incorporated herein by reference). Yet additional exemplary mutations may be engineered in the N gene start sequence, for example by changing one or more nucleotides in the N gene start sequence, e.g., at a position corresponding to nucleotide 62 of JS cp45.
From JS cp45 and other biologically derived PIV mutants, a large xe2x80x9cmenuxe2x80x9d of attenuating mutations is provided, each of which mutations can each be combined with any other mutation(s) for adjusting the level of attenuation in a recombinant PIV bearing C, D, and/or V deletion or knock out mutation(s). For example, mutations within recombinant PIVs of the invention include one or more, and preferably two or more, mutations of JS cp45. Desired C, D, and/or V deletion or knock out mutants of the invention selected for vaccine use often have at least two and sometimes three or more attenuating mutations to achieve a satisfactory level of attenuation for broad clinical use. Preferably, recombinant PIV bearing C, D, and/or V deletion or knock out mutation(s) incorporates one or more attenuating mutation(s) stabilized by multiple nucleotide substitutions in a codon specifying the mutation.
Additional mutations which can be adopted or transferred to C, D, and/or V deletion or knock out mutants of the invention may be identified in non-PIV nonsegmented negative stranded RNA viruses and incorporated in PIV mutants of the invention. This is readily accomplished by mapping the mutation identified in a heterologous negative stranded RNA virus to a corresponding, homologous site in a recipient PIV genome or antigenome and mutating the existing sequence in the recipient to the mutant genotype (either by an identical or conservative mutation), as described in U.S. Provisional Application No. 60/129,006, filed by Murphy et al. on Apr. 13, 1999, incorporated herein by reference.
In addition to the above described mutations, infectious C, D, and/or V deletion or knock out mutants desirably incorporate heterologous, coding or non-coding nucleotide sequences from any heterologous PIV or PIV-like virus, e.g., HPIV1, HPIV2, HPIV3, bovine PIV (BPIV) or murine PIV (MPIV) to form a chimeric genome or antigenome. For example, recombinant PIV of the invention may incorporate sequences from two or more wild-type or mutant PIV strains, e.g., HPIV1 and HPIV3. Alternatively, C, D, and/or V deletion or knock out mutants of the invention may incorporate sequences from a human and non-human PIV, e.g., HPIV and BPIV. Preferably, one or more human PIV coding or non-coding polynucleotides in a xe2x80x9crecipientxe2x80x9d or xe2x80x9cbackgroundxe2x80x9d genome or antigenome are substituted with a counterpart sequence from a heterologous PIV or non-PIV virus, alone or in combination with one or more selected attenuating point mutations, e.g., cp and/or ts mutations, to yield novel attenuated vaccine strains. The isolated infectious PIV particle is preferably a human PIV, more preferably human PIV3 (HPIV3) (see, e.g., U.S. Provisional Application No. 60/143,134, filed by Bailey et al. on Jul. 9, 1999, incorporated herein by reference).
In related aspects of the invention, isolated, infectious PIV particles are provided which incorporate nucleotide sequences from HPIV3 joined to at least one sequence from a heterologous PIV, such as HPIV1, HPIV2, BPIV or MPIV. For example, entire genes of HPIV3 may be replaced by counterpart genes from other forms of PIV, such as the HN and/or F glycoprotein genes of PIV1 or PIV2. Alternatively, a selected genome segment, for example a cytoplasmic tail, transmembrane domain or ectodomain of HN or F of HPIV1 or HPIV2, can be substituted for a corresponding gene segment in a counterpart HPIV3 gene to yield constructs encoding chimeric proteins, e.g. fusion proteins having a cytoplasmic tail and/or transmembrane domain of PIV3 fused to an ectodomain of PIV1 or PIV2. Alternatively, genes or genome segments from one PIV can be added (i.e., without substitution) within a heterologous PIV background to create novel immunogenic properties within the resultant clone.
In addition to recombinant PIV having C, D, and/or V deletion or knock out mutations, the invention provides related cDNA clones, vectors and particles, each of which incorporate one or more of the subject, phenotype-specific mutations set forth herein. These are introduced in selected combinations, e.g., into an isolated polynucleotide which is a recombinant cDNA genome or antigenome, to produce a suitably attenuated, infectious virus or subviral particle upon expression, according to the methods described herein. This process, coupled with routine phenotypic evaluation, provides C, D, and/or V deletion or knock out mutants having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, genetic stability, etc. In particular, vaccine candidates are selected which are attenuated and yet are sufficiently immunogenic to elicit a protective immune response in the vaccinated mammalian host.
In yet additional aspects of the invention, C, D, and/or V deletion or knock out mutants, with or without additional attenuating mutations adopted, e.g., from a biologically derived mutant virus, are constructed to have additional nucleotide modification(s) to yield a desired phenotypic, structural, or functional change. Typically, the selected nucleotide modification will specify a phenotypic change, for example a change in growth characteristics, attenuation, temperature-sensitivity, cold-adaptation, plaque size, host range restriction, or immunogenicity. Structural changes in this context include introduction or ablation of restriction sites into PIV-encoding cDNAs for ease of manipulation and identification.
In preferred embodiments, nucleotide changes within the genome or antigenome of a C, D, and/or V deletion or knock out mutant include modification of an additional viral gene by partial or complete deletion of the gene or reduction or ablation (knock out) of its expression. Target genes for mutation in this context include the nucleocapsid protein N, phosphoprotein P, large polymerase subunit L, matrix protein M, hemagglutinin-neuraminidase protein HN, fusion protein F. To the extent that the recombinant virus remains viable and infectious, each of these proteins can be selectively deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to achieve novel C, D, and/or V deletion or knock out mutants. For example, one or more of these genes, may be deleted in whole or in part or its expression reduced or ablated (e.g., by introduction of a stop codon, by a mutation in an RNA editing site, by a mutation that alters the amino acid specified by an initiation codon, or by a frame shift mutation) to alter the phenotype of the resultant recombinant clone to improve growth, attenuation, immunogenicity or other desired phenotypic characteristics.
Alternative nucleotide modifications in C, D, and/or V deletion or knock out mutants of the invention include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected gene in the recombinant genome or antigenome. In one example, a cis-acting regulatory sequence of one PIV gene is changed to correspond to a heterologous regulatory sequence, which may be a counterpart cis-acting regulatory sequence of the same gene in a different PIV, or a cis-acting regulatory sequence of a different PIV gene. For example, a gene end signal may be modified by conversion or substitution to a gene end signal of a different gene in the same PIV strain. In other embodiments, the nucleotide modification may comprise an insertion, deletion, substitution, or rearrangement of a translational start site within the recombinant genome or antigenome, e.g., to ablate an alternative translational start site for a selected form of a protein.
In addition, a variety of other genetic alterations can be produced in a PIV genome or antigenome having a deletion or knock out of C, D, and/or V, alone or together with one or more attenuating mutations adopted from a biologically derived mutant PIV. For example, genes or genome segments from non-PIV sources may be inserted in whole or in part. Alternatively, the order of genes can be changed or a PIV genome promoter can be replaced with its antigenome counterpart. Different or additional modifications in the recombinant genome or antigenome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions or elsewhere. Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
In yet additional aspects, polynucleotide molecules or vectors encoding the recombinant PIV genome or antigenome can be modified to encode non-PIV sequences, e.g., a cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen (e.g., virus, bacterium or fungus) capable of eliciting a protective immune response in an intended host. In one such embodiment, C, D, and/or V deletion and knock out mutants are constructed that incorporate a gene or genome segment from a respiratory syncytial virus (RSV), for example a gene encoding an antigenic protein (e.g., an F or G protein), immunogenic domain or epitope of RSV.
In related aspects of the invention, compositions (e.g., isolated polynucleotides and vectors incorporating a PIV-encoding cDNA) and methods are provided for producing an isolated infectious recombinant PIV bearing a C, D, and/or V deletion or knock out mutation(s). Included within these aspects of the invention are novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a PIV genome or antigenome which is modified by a partial or complete deletion of the C, D, and/or V ORF(s), or one or more nucleotide changes that reduce or ablate expression thereof. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules encoding N, P, and L proteins. These proteins also can be expressed directly from the genome or antigenome CDNA. The vector(s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious C, D, and/or V deletion or knock out mutant PIV particle or subviral particle.
The above methods and compositions for producing C, D, and/or V deletion and knock out mutant PIV yield infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the authentic PIV virus particle and is infectious as is. It can directly infect fresh cells. An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic RNA and the N, P, and L proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells. Subviral particles provided within the invention include viral particles which lack one or more protein(s), protein segment(s), or other viral component(s).
In other embodiments the invention provides a cell or cell-free lysate containing an expression vector which comprises an isolated polynucleotide molecule comprising a C, D, and/or V deletion or knock out mutant PIV genome or antigenome as described above, and an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, and L proteins of PIV. One or more of these proteins also can be expressed from the genome or antigenome cDNA. Upon expression the genome or antigenome and N, P and L combine to produce an infectious PIV virus or subviral particle.
In other embodiments of the invention a cell or cell-free expression system (e.g., a cell-free lysate) is provided which incorporates an expression vector comprising an isolated polynucleotide molecule encoding a PIV genome or antigenome bearing a C, D, and/or V knockout mutation(s), and an expression vector comprising one or more isolated polynucleotide molecules encoding N, P, and L proteins of a PIV. Upon expression, the genome or antigenome and N, P, and L proteins combine to produce an infectious PIV particle, such as a viral or subviral particle.
The recombinant PIVs of the invention are useful in various compositions to generate a desired immune response against PIV in a host susceptible to PIV infection. Attenuated C, D, and/or V deletion and knock out mutants of the invention are capable of eliciting a protective immune response in an infected mammalian host, yet are sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host. The attenuated virus or subviral particle may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified. The virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.
The invention further provides novel vaccines comprising a physiologically acceptable carrier and/or adjuvant and an isolated attenuated C, D, and/or V deletion or knock out mutant PIV particle or subviral particle. In preferred embodiments, the vaccine is comprised of a C, D, and/or V deletion or knock out mutant PIV having at least one, and preferably two or more additional mutations or other nucleotide modifications as described above to achieve a suitable balance of attenuation and immunogenicity. The vaccine can be formulated in a dose of 103 to 107 PFU of attenuated virus. The vaccine may comprise attenuated C, D, and/or V deletion or knock out virus that elicits an immune response against a single PIV strain or against multiple PIV strains. In this regard, C, D, and/or V deletion and knock out mutant PIV can be combined in vaccine formulations with other PIV vaccine strains, or with other viral vaccine viruses such as an RSV.
In related aspects, the invention provides a method for stimulating the immune system of an individual to elicit an immune response against PIV in a mammalian subject. The method comprises administering a formulation of an immunologically sufficient amount of an attenuated, C, D, and/or V deletion or knock out mutant PIV in a physiologically acceptable carrier and/or adjuvant. In one embodiment, the immunogenic composition is a vaccine comprised of a C, D, and/or V deletion or knock out mutant PIV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications specifying a desired phenotype as described above. The vaccine can be formulated in a dose of 103 to 107 PFU of attenuated virus. The vaccine may comprise attenuated C, D, and/or V deletion or knock out mutant PIV virus that elicits an immune response against a single PIV, against multiple PIVs, e.g., HPIV1 and HPIV3, or against one or more PIV(s) and a non-PIV pathogen such as RSV. In this context, C, D, and/or V deletion and knock out mutant PIV can elicit a monospecific immune response or a polyspecific immune response against multiple PIVs, or against one or more PIV(s) and a non-PIV pathogen such as RSV. Alternatively, C, D, and/or V deletion and knock out mutant PIV having different immunogenic characteristics can be combined in a vaccine mixture or administered separately in a coordinated treatment protocol to elicit more effective protection against one PIV, against multiple PIVs, or against one or more PIV(s) and a non-PIV pathogen such as RSV. Preferably the immunogenic composition is administered to the upper respiratory tract, e.g., by spray, droplet or aerosol. Preferably the immunogenic composition is administered to the upper respiratory tract, e.g., by spray, droplet or aerosol.